Acanthamoeba myosin-II minifilaments assemble on a millisecond time scale with rate constants greater than those expected for a diffusion limited reaction.
We have shown previously that Acanthamoeba myosin-II minifilaments assemble by three successive dimerization steps, forming, progressively, monomers, antiparallel dimers, antiparallel tetramers, and finally the full size octameric minifilament (Sinard, J. H., Stafford, W. F., and Pollard, T. D. (1989) J. Cell Biol. 109, 1537-1548). In the current study, we investigate the kinetics of the assembly of these minifilaments, initiating assembly by the rapid dilution of salt in a stopped-flow light scattering apparatus. The majority of the reaction is completed within 50 ms and is greater than 90% completed within 1 s. Assembly data over a greater than 6-fold myosin concentration range can be fit using the successive dimerization mechanism with a single set of rate constants. Second order rate constants for the initial steps in the assembly reaction exceed 10(8) M-1 s-1, and equilibrium dissociation constants predict a very low critical concentration, consistent with previous data. Other possible assembly mechanisms do not adequately fit all of the available data. Filament disassembly at 300 mM KCl is even more rapid, and there is both an increase in the dissociation rate constants and a decrease in the association rate constants with increasing KCl. Aggregation of minifilaments induced by Mg2+ is much slower and takes many minutes to reach equilibrium.
Sinard
J H
JH
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Pollard
T D
TD
D000048:Acanthamoeba / Q000378:metabolism*; D000818:Animals; D003599:Cytoskeleton / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D009218:Myosins / Q000378:metabolism*; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D012542:Scattering, Radiation; D013997:Time Factors
D046911:Macromolecular Substances; D011189:Potassium Chloride; D009218:Myosins; D008274:Magnesium